Three broadly connected and important neurotransmitter systems in the central nervous system are notably influenced by gonadal steroids. Centrifugal afferents from these systems furnish significant inputs to the olfactory bulb (OB), which affords a very accessible approach for study of these systems. These systems are cholinergic from the diagonal band nuclear complex (DB), noradrenergic, from the locus ceruleus (LC), and serotonergic from the raphe nuclei (RN). These nuclei appear to be the sole source of these transmitters in the OB. Each nuclear area has been found to contain a contingent of target neurons for gonadal steroids. This proposal is designed to test the hypothesis that the steroid sequestering neurons in the DB, LC and RN exert significant influence on the development and function of their major transmitter projections to the olfactory bulb, probably in a sexually dimorphic way. We will start by using adult rats of both sexes to quantify the numerical relationship between the projections of these nuclei to the OB and their respective number of steroid target neurons, using a double labelling protocol of retrograde fluorescent tracers and autoradiographic demonstration of trget neurons for either estradiol (3H-E2) or dihydrotestosterone (3H-DHT). On top of this double labelling, an immunocytochemical label will be added to some preparations in order to verify the neurotransmitter type of the labelled neurons. A parallel series of experiments will be done in which male and female rats will be castrated as neonates after which studies will be done to assess the effects of this on the projections of the DB, LC or RN to the OB. The assessment of effects will include quantitation of projections by microscopic and biochemical techniques, evaluation of specific transmitter changes and quantification of neurotransmitter receptors with radioligand binding. Any changes will be used to evaluate later studies on the effects of hormone replacement. The quantitation and analysis of our microscopic data will be made possible by a unique, powerful image analysis system with an image intensifier and computer analysis. Long term, this study will have great significance for better understanding of the way hormones can influence the major tone-setting systems of the CNS, with considerable relevance for better understanding of mental health and senile dementia.